The L. infantum antigens were provided by the parasitology department of the School of Public Health at Tehran University of Medical Sciences. The main procedure for the preparation of DAT antigen was mass cultivation of promastigotes of L. infantum LON‐49 in RPMI1640, parasite trypsinization, Coomassie blue staining and fixing with 2% formaldehyde (el Harith et al., 1989). The dog sera samples were evaluated by DAT based on the procedure originally described by Harith et al. (el Harith et al., 1989) and utilized in several studies in Iran (Mohebali et al., 2006; Sarkari et al., 2015). A cut‐off titre of ≥1:320 was considered positive (Mohebali et al., 2005).

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